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Subjects

Abstract

A short, 14-amino-acid segment called SP1, located in the Gag structural protein1, has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors4,5. Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP6) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1.

Acknowledgements

We thank J. Briggs for discussions and reading of the manuscript. This work was supported by the National Institutes of Health (NIH) grants R01-GM107013 (V.M.V.), R01-GM105684 (G. W. Feigenson), P30-GM110758 and P50-GM082251 (J.R.P.), R01-AI129678 (O.P. and B.K.G.-P.), U54-GM103297 (O.P.), and R01-GM110776 (M.C.J.). F.K.M.S. was supported by Deutsche Forschungsgemeinschaft grant BR 3635/2-1 awarded to J. A. G. Briggs. J.M.W. was supported by NIH postdoctoral fellowship grant F32-GM115007. Anton computer time was provided by the Pittsburgh Supercomputing Center (PSC) through NIH grant R01-GM116961. The Anton machine at PSC was generously made available by D. E. Shaw Research. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation (NSF) grant number OCI-1053575. Specifically, it used the Bridges system, which is supported at PSC by NSF award number ACI-1445606. Some of The EM work was conducted at the Molecular Electron Microscopy Core facility at the University of Virginia.

Reviewer information

Nature thanks E. Freed and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Contributions

R.A.D. performed protein purification and in vitro assembly. F.K.M.S. did comparative analyses of cryo-EM and crystal structure data. K.K.Z, J.M.W., B.K.G.-P. and O.P. carried out crystallization trials and structure determination. B.K.G.-P. performed 2D cryo-EM. J.R.P. and C.X. performed all-atom MD simulations. T.D.L., C.L.R. and M.C.J., performed cell biology and virology. The manuscript was written primarily by R.A.D., J.R.P., B.K.G.-P., O.P. and V.M.V. The project was originally conceived by R.A.D., with input from all authors throughout experimentation and manuscript preparation.

a, The crystal structure of CACTDSP1 bound to IP6 (cyan) was superimposed on a previously described model of the CA-SP1 segment build into cryo-EM densities of immature HIV-1 particles (PDB 5L93, orange2). Note the close correspondence in K359 rotamers, which were modelled independently in the two structures. For visualization purposes, only one of the six possible IP6 conformations is displayed. b, RMSD calculations of the crystal structure and PDB 5L93. For full-length (residues 149–237) and CA-SP1 (residues 223–237), the RMSDs were calculated only for the atoms that were modelled in both maps. If a sidechain was not modelled, the entire residue was omitted from the calculation. The overall agreement of the models is very high, indicating that the crystal structure corresponds well with conformations found in the virus. c, The CACTDSP1 bound to IP6 (orange and red, respectively) was fitted into two previously published cryo-EM densities2 from VLPs collected from cells (EMD-2706 and EMD-4017). Both maps are shown at 8.8 Å, which is the resolution of the lower resolved map, EMD-2706. In the zoomed insets, only the density corresponding to IP6 is shown. Matching of models and maps and RMSD calculations were performed in Chimera.

a, Top and side views of the unbiased mFo–DFc difference density (blue mesh, 2σ) ascribed to the bound IP6. Shown are six IP6 molecules docked in six rotationally equivalent positions, consistent with the six-fold rotational symmetric density. b, Top view of the docked IP6 molecules within the CACTDSP1 hexamer. Unbiased mFo–DFc difference densities (blue mesh) are also shown for both the bound IP6 and sidechains of Lys290 (green) and Lys359 (cyan). Density for Lys359 is more pronounced, which we interpret to mean that this residue adopts a more restricted range of rotamers for binding IP6.

a, Structural changes observed after 2 μs of molecular dynamics simulations of CACTDSP1 with and without bound IP6. b, RMSDs of the ligand-bound and unbound forms of the CACTDSP1 hexamer. c, RMSFs of the central hexamer during the simulation. The RMSF was averaged over the six central monomers; dashed line shows the s.d. for each residue.

Supplementary information

FACs gating strategy. a, Events were plotted along forward scatter (FSC) and side scatter (SSC) axises using FlowJo. Events with the right morphology were gated as "Live" cells and the position of the gate was copied onto all samples. b, Events from the "Live" gate were isolated and plotted along GFP and RFP intensity axes. The "Non-Fluorescent" gate was created based on a HEK293FT fluorescence negative sample (plot not shown) and the gate was copied onto all isolated "Live" samples. The "GFP-Positive" gate was created based on a HEK293FT GFP positive sample (plot not shown) and the gate was copied onto all isolated "Live" samples. Representative comparison of two cell types transduced with HIV Env deficient virus with a GFP reporter (HEK293FT = WT and IPPK KO = HEK293FT with inositol-pentakisphosphate 2-Kinase knocked out).